Store-operated Ca2+ entry (SOCE) underlies numerous cellular processes throughout the body and initiates signaling cascades in T lymphocytes and microglia that cause changes in motility, secretion of cytolytic granules, cytokine release, and cell proliferation. The channels that underlie SOCE have been identified recently through RNA interference (RNAi) screening as a conserved family of four transmembrane-spanning proteins named Orai that are activated by STIM proteins in the ER membrane. Isoforms of these proteins are expressed throughout the body in a tissue-specific manner. Important cellular functions of Orai1 have been identified in lymphocytes, microglia, mast cells, blood platelets, sweat and salivary glands, dentition, vascular smooth muscle, endothelial cells, and skeletal muscle. In the immune system, STIM1 and Orai1 mediate antigen-induced Ca2+ signaling, motility inhibition at the site of antigen presentation, secretion of cytolytic granules by CD8+ T cells and NK cells, and gene expression responses that lead to cytokine release and cell proliferation. More recent studies show that SOCE is a major route of Ca2+ influx in microglia. Additionally STIM1 and Orai1 play a role in mediating functional Ca2+ responses to purinergic P2Y activation including chemotaxis and phagocytosis. STIM and Orai proteins are being developed as targets for treatment of autoimmune diseases and prevention of transplant rejection. Our overall goal is to understand how Orai channels function at the molecular and cellular level. Orai channels in the plasma membrane are unrelated to other known ion channels and have unusual characteristics that distinguish them, including a very high degree of selectivity for Ca2+, low single-channel conductance, and activation by binding of a small cytosolic domain of the STIM protein. Moreover, the human Orai1 and Orai3 proteins differ in their activation requirements and tissue distribution. In this project, we have three goals. We seek to understand: 1) how Orai1 is activated by STIM1 and interacts with adjacent Orai1 channels in puncta to generate localized Ca2+ signals; 2) how local Ca2+ signals modulate T cell motility, turning behavior and stopping during immune surveillance; and 3) how Orai1 regulates Ca2+ signaling network that underlie the complex motility patterns seen in microglia. To accomplish these Aims, we have developed and continue to develop new tools for monitoring local Ca2+ signals that will be broadly applicable. Our studies will include electrophysiological analysis of gating and ion permeation, optical imaging of Ca2+ flux through Orai1 channels, and two-photon imaging of in situ cellular motility. Overall, our project will provide fundamental insights into the Orai1 proteins that are currently being targeted for treatment of autoimmune disorders, chronic inflammatory conditions, and neurodegerative diseases such as Alzheimer?s.